The present invention relates generally to electrospray ionization for mass spectrometry, and more particularly the invention relates to an apparatus and method for producing an electrospray from a sample solution for introduction into mass spectrometer.
Mass spectrometry is an important tool in the analysis of a wide range of chemical compounds. Specifically, mass spectrometers can be used to determine the molecular weight of sample compounds. The analysis of samples by mass spectrometry consists of three main stepsxe2x80x94formation of gas phase ions from sample material, mass analysis of the ions to separate the ions from one another according to ion mass, and detection of the ions. A variety of means exist in the field of mass spectrometry to perform each of these three functions. The particular combination of means used in a given spectrometer determine the characteristics of that spectrometer.
The present invention relates to the first of these steps the formation of gas phase ions from a sample material. More particularly, the present invention relates to electrospray ionization (ESI), one such means for producing gas phase ions from a sample material. Electrospray ionization, was first suggested by Dole et al. (M. Dole, L. L. Mack, R. L. Hines, R. C. Mobley, L. D. Ferguson, M. B. Alice, J. Chem. Phys. 49, 2240, 1968). Generally, in the electrospray technique, analyte is dissolved in a liquid solution and sprayed from a needle. The spray is induced by the application of a potential difference between the tip of the needle and a counter electrode. Specifically, a voltage of several kilovolts is applied between, for example, a metal capillary and a flush surface separated by a distance of approximately 20 to 50 millimeters. Under the effect of the electric field, a liquid in the capillary is dielectrically polarized at the end of the capillary. The liquid is then pulled out into a cone, known as the Taylor cone. The surface tension of the liquid at the pointed end of the cone is no longer able to withstand the attraction of the electric field, and this causes a small electrically charged droplet to be detached. The charged droplet flies with great acceleration to the flush counter electrode, effected by the inhomogeneous electric field. During the flight of the liquid, evaporation occurs and the droplets are slowed down. The spray results in the formation of finely charged droplets of solution containing analyte molecules. The larger ions become ionized, and move towards the counter electrode to be transferred into the vacuum system of a mass spectrometer, for example, through a narrow aperture or capillary very large ions can be formed in this way. For example, ions as large as 1 MDa have been detected by ESI in conjunction with mass spectrometry (ESMS).
Electrospray, as in the present invention, facilitates the formation of ions from sample material. It should be noted that the size of the droplets produced in the ESI technique is dependant upon the size of the sprayer used. The terms nanospray or micro spray are used to indicate the use of very small sprayers in electrospray technique. In other words, a sprayer having an opening of less than about 10 xcexcm (microns) will produce a nanospray, a sprayer having an opening of between approximately 10-100 xcexcm (microns) will produce a micro spray, and a sprayer having an opening of greater than 100 xcexcm (microns) will produce an electrospray. For convenience, all three are referred to generally as xe2x80x9celectrospray,xe2x80x9d in as much as the present invention can be used with each.
Referring to FIG. 1, depicted is an ionization source of co-pending application Ser. No. 09/570,797 which shows an API source for generating ions from a sample for subsequent analysis. As shown, the ionization source 101 comprises spray chamber 1, transfer region 2, first pumping region 5, second pumping region 4, hinge 9, flange 10, and source block 16. During normal operation of the ionization source 101 incorporating an ESI source it is anticipated that numerous other elements may be used within ionization source 101 as shown in FIG. 1. These may include vacuum pump 15, ion transfer devices such as capillary 6 having an entrance end 7, and exit end 19 and inner channel 8, multipole devices such as pre-hexapole 11 and hexapole 12, as well as other ion optic devices such as skimmers 13 and 14 and exit electrodes 17.
Initially, sample solution is formed into droplets at atmospheric pressure by spraying the sample solution from a spray needle 20 into spray chamber 1. The spray may be induced by the application of a high potential between the tip of spray needle 20 and the capillary entrance end 7 within spray chamber 1. Then, these sample droplets evaporate while in the spray chamber 1 thereby leaving behind sample ions. These sample ions are accelerated or directed toward capillary entrance 7 and into channel 8 by the electric field generated between spray needle 20 and capillary entrance 7. These ions are then transported through capillary 6 to capillary exit 19, due to the flow of gas created by the pressure differential between spray chamber 1 and first transfer region 2.
The present invention relates particularly to the sprayers used within electrospray ionization. Presently, known electrospraying techniques teach that it is necessary to take active steps to ionize the solution for analysis in the mass spectrometer. For instance, FIG. 2 depicts a typical prior art electrospray needle 21. As shown, needle 21 comprises an elongated capillary structure tapered at one end to form tip 22. Needle 21 includes a plenum 24 to receive the liquid sample. Plenum 24 is shown having an interval region larger than that of the capillary section of needle 21. Liquid sample flows from plenum 24 through upstream inlet 25 into the capillary section of ejection through tip 26. Plenum 24 may be electrically conductive so that a voltage applied to the plenum 24 will allow for the transfer of charge into the liquid stream. Alternatively, a charge can be imposed on the capillary section of needle 21. The applied voltage produces an electrical field which is arranged such that it is at its highest at the tip 26 such that the charge and field at tip 26 are high enough to form the electrospray (i.e. charged droplets) . Such a prior art apparatus consists only of a single needle which, is a very thin capillary, producing flow rates on the order of 20 nL/min. Further, such a needle must be loaded through its back end (i.e. the plenum 24, as shown in FIG. 2), not through the tip 25. This can be a very time consuming process.
Typically, nanospray needles are produced by taking a glass capillary having a relatively large diameter and pulling and/or machining it to a tip. Then a metal coating is vapor deposited onto its outer surface, as disclosed in Mann U.S. Pat. No. 5,504,329 (Mann). The needle shown in FIG. 3 is the result of such a process. Needles such as this are formed by using heat to soften glass capillary tubing and pulling the tip end to form the needle""s tapered tip 27. These needles are generally single use, and must be loaded with sample solution using micropipettes or some other means for loading sample solution through the end 28 of the needlexe2x80x94the end opposite the spray tipxe2x80x94using a micropipette.
Such needles are generally single use, and require the sample to be reloaded through its back end after each use. The prior art needles breed inaccuracy because the conditions have to be replicated with each removal and replacement. In addition, the fragile nature of the needles, combined with their limited use, makes replacement costs a significant expense for their users. Also, because these needles are extremely fragile, replacement is frequent, which is both costly and time consuming.
Once these prior art needles are formed, a means of making electrical contact is required. Prior art needles have been made from small metal tubing (e.g., a steel syringe needle) or dielectric tubing (e.g., glass, fused silica or polymer tubing). If the needle is made of an insulating material, there are generally three ways that the prior art teaches to make a needle capable of electrical contact: (i) applying thin metal films directly onto the dielectric tubing, (ii) supporting the dielectric tip inside a secondary metal tube that contacts the liquid as it exits the dielectric tubing and (iii) making a direct electric contact with the solution from a remote position. The most commonly used of these is the application of a thin metal film (e.g., gold or platinum) directly onto the dielectric tubing.
However, due to their relatively inert nature, such metals often show poor adhesion to the substrate materials, which reduces ESI stability and eventually leads to ESI tip failure. As the analyte is sprayed from the tip, the metal coating can rapidly deteriorate through peeling or flaking. An attempted solution to this problem has been to apply an interlayer material, such as chromium or sulfur containing silanes; which adheres to both the metal and the substrate. However, this has not entirely solved the problem because such interlayer materials are subject to chemical attack (i.e., dissolution, in the case of chromium, or bond cleavage, in the case of silanes).
Valaskovic U.S. Pat. No. 5,788,166 (Valaskovic), for example, uses a process of applying a metal overcoating on a dielectric capillary needle. The capillary needle is constructed by heating fused-silica tubing with a laser, then pulling the tube until its internal diameter is in the range of 3 xcexcm. The pulling process is followed by chemical etching and surface metallization. The pulling results in formation of slowly tapered capillary edges and a tip having a very small inner diameter. The chemical etching process forms the tapered outer wall and a sharp point at the tip of the needle. The surface metallization applies a thin metal contact layer on the outer wall of the needle, to allow for electrical contact. Then an electrically insulating overcoat is applied. The overcoat essentially fixes the conductive metal contact layer into place, although the electrically insulating overcoat does not improve the adhesion of the metal to the capillary.
Because the pulling process is used on fused silica tubing, the extra step of metallization is required. The pulling process results in slowly tapered edges, which culminate in a sharp point. This point is then etched to create a narrow diameter opening at the distal end (or tip) of the pulled tubing (i.e., forming a needle). A needle such as this has the disadvantage of the formation of xe2x80x9cbubblesxe2x80x9d in the solution within the needle, which interferes with the spray of the solutionxe2x80x94in fact, it may even stop flow of the solution from the needle. In other words, having such a narrow diameter at the distal end (or tip) of the needle permits air pockets to form at the base of the tip. That is, solution near the distal end may begin to evaporate, thereby forming air pockets. These air pockets then permeate through the solution toward the proximal end (due to the larger space available), effectively xe2x80x9cblockingxe2x80x9d the spray of solution from the needle. The glass structure of the needle also contributes to the formation of these air pockets, as the solution is held within the needle due to capillary action. In other words, the solution grips the inner surface of the needle as the air pockets permeate through the interior of the needle.
Other forms of electrospray include pneumatic assisted, thermal assisted, or ultrasonic assisted, or the addition of arc suppression gases so that higher voltages can be applied during electrospray formation. Pneumatically assisted sprayers typically have a much larger tip (greater than 100 xcexcm) than, for example, nanosprayers (around 5 xcexcm) (See FIG. 4 for an example of a nanospray needle). When using pneumatically assisted sprayers, sample solution is typically pumped (for example, via a syringe pump) into the sprayer. Sample aliquots can then be injected into this solution stream either manually or automatically (i.e., by a robot or other machine). However, the conventional process of injecting sample into sprayers by machines is cumbersome, as the process is difficult to control. That is, filling the needle through its proximal end is not practicalxe2x80x94since the opening at the proximal end is so small. The glass capillary, with the opening at the end, provides a measure of resistance during filling, and therefore must be performed carefully with a micropipette.
Accordingly, prior to the present invention, a need has existed for a multiple use, robust, spray needle and sprayer having a geometry that eases the elimination of voids or bubbles. It is a purpose of the invention to provide such a spray needle and sprayer, as well as a method of operating a mass spectrometer using a spray needle and sprayer to produce an electrospray formed from a sample solution. It is also a purpose of the present invention to provide a means and method of operating a mass spectrometer which utilizes the apparatus with a variety of ionization techniques (i.e., ESI, MALDI, etc.)
One aspect of the present invention is to provide an apparatus and method of facilitating the introduction of a liquid sample into a mass spectrometer for subsequent analysis. To address the foregoing problems, the present invention provides a sprayer which is reusable, robust, and easy to load. Furthermore, the present invention provides a spray needle and sprayer which has a geometry that minimizes the formation of voids or bubbles, thereby providing improved results in the analysis of the sample solution, as demonstrated in the mass spectra of FIG. 10 obtained in a mass analysis performed using the spray needle according to the preferred embodiment disclosed herein.
Specifically, one embodiment of the present invention comprises a two component spray needle (i.e., a support and a tip). Advantages of a spray needle having this configuration include ease of sample loading, minimization of bubble formation or voids, durability, reusability, ease of automation, ease of replacement, increased reproduction of analysis results, etc. For example, if after repeated uses the tip is no longer functional, a new tip may be constructed, and attached to the intact support.
Another embodiment of the present invention provides a single component spray needle and sprayer having an opening along its length to facilitate the introduction or loading of a sample solution into the needle. In other words, the spray needle can be filled with the solution through its an elongated slit along its length by merely dipping the needle into the sample solution. This allows for the liquid to be drawn in through the tip into the body of the spray needle via capillary action. At the same time, this may limit the droplet size upon ejection of the sample from the needle. The opening also provides for unique spraying capabilities due to its geometry and length. Furthermore, because the spray needle does not need to be loaded via the rear opening (or proximal end), the spray needle can be easily employed within automated systems.
Yet another embodiment of the present invention comprises a single unit spray needle having a slit along its length as well as having the tip end diagonally cut (as shown in FIGS. 7A-7C). The construction of this embodiment provides a robust needle which facilitates the introduction of sample solution into the spray needle through its proximal end (or tip) as well as facilitates the production of very small sample droplets for ionization. In addition, the spray needle of this embodiment can be loaded through a dipping process, making it ideal for use with an automated process. The needle of this embodiment also minimizes the formation of bubbles or voids in the sample solution.
Yet a further embodiment of the invention comprises a multitip spray needle (as shown in FIGS. 11A-C). Such a spray needle preferably embodies the structure of the preferred embodiment shown in FIGS. 5A-C, but alternatively, may embody the alternative structures shown in FIGS. 6A-C and 7A-C. Specifically, a multi-tip spray needle according to the invention may comprise a plurality of (e.g., 20, 50, 100, etc.) very fine (i.e., on the order of 50 xcexcm or less) elements at the needle""s distal end. An advantage of such a multi-tip structure is the facilitation of the spray of extremely fine droplets, having the effect of maximizing the introduction of sample ions into the mass analyzer from the source region.
Other objects, features, and characteristics of the present invention, as well as the methods of operation and functions of the related elements of the structure, and the combination of parts and economies of manufacture, will become more apparent upon consideration of the following detailed description with reference to the accompanying drawings, all of which form a part of this specification.
A further understanding of the present invention can be obtained by reference to a preferred embodiment set forth in the illustrations of the accompanying drawings. Although the illustrated embodiment is merely exemplary of systems for carrying out the present invention, both the organization and method of operation of the invention, in general, together with further objectives and advantages thereof, may be more easily understood by reference to the drawings and the following description. The drawings are not intended to limit the scope of this invention, which is set forth with particularity in the claims as appended or as subsequently amended, but merely to clarify and exemplify the invention.